This project's long-term goal is the development and clinical validation of a useful diagnostic blood test for the disease sarcoidosis. Sarcoidosis is an inflammatory disorder characterized by granulomas, most commonly in the lungs. Its cause is unknown. The diagnosis of sarcoidosis is currently one of exclusion;there is no reliable, affirmative, rule-in, diagnostic test. Namely, the patient<s symptoms often result in a chest x-ray, which leads to a biopsy showing non-caseating granulomas. Other potential causes, such as TB or exposure to beryllium, are excluded, thereby leading to a diagnosis of sarcoidosis. Our goal is a blood diagnostic test that affirmatively indicates sarcoidosis. This project is also a first reduction to practice for a newly developed discovery platform for identifying new serologic assay targets. The principle of the approach is that inflammatory disorders cause the production of serum antibodies. Identifying these disease-associated antibodies (in diseases of unknown etiology) could be of enormous value. The previously difficult hurdle has been that characterization of antibody immunoreactivity requires the antigen. In sarcoidosis, the antigen is unknown. Consequently, we used a random peptide combinatorial library displayed on bacteriophage in lieu of antigen. We established a method for identifying peptides (from the library) that are immunoreactive with patient antibodies but not with serum antibodies from a reference group. This kind of differential library enrichment using polyclonal serum antibodies has previously been challenging. There are two Specific Aims: (1) Sensitivity &specificity analysis of the candidate peptide targets (displayed on phage), and (2) Final assay design validation. In Aim 1, we will test the (>100 already identified) candidate peptide phage, to determine their sensitivity and specificity in the diagnosis of sarcoidosis. Our Preliminary Data shows that these peptides are immunoreactive with at least some sarcoidosis sera, and not with normal control sera or sera from PPD+ individuals. We will analyze the immunoreactivity of each peptide with at least 58 sarcoidosis sera and >110 control sera. The sera are provided through a consortium of clinical collaborators at Tufts Medical Center, Boston University Medical Center, and Vanderbilt University Medical Center. In Aim 2, we will combine the best- performing peptides into a single immunoassay, for superior sensitivity. In addition to sensitivity and specificity, we will also evaluate precision, linearity, and assay stability. In a future Phase II, we will conduct a prospective study, with an independent set of patients, for regulatory submission. PUBLIC HEALTH RELEVANCE: This project is for the purpose of creating a blood test for sarcoidosis, a disease of unknown cause and for which there is no definitive test. The new technology that we employed to discover the immune response targets for this test is applicable to many other diseases involving the body<s immune system.